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- EMDB-12213: CryoEM structure of disease related M854K MDA5-dsRNA filament in ... -

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Basic information

Entry
Database: EMDB / ID: EMD-12213
TitleCryoEM structure of disease related M854K MDA5-dsRNA filament in complex with ATP
Map data
Sample
  • Complex: M854K MDA5-dsRNA filament in complex with ATP
    • Complex: INTERFERON-INDUCED HELICASE C DOMAIN-CONTAINING PROTEIN
      • Protein or peptide: Interferon-induced helicase C domain-containing protein 1
    • Complex: RNA
      • RNA: RNA (5'-R(P*CP*UP*CP*UP*CP*CP*UP*CP*GP*GP*CP*UP*UP*G)-3')
      • RNA: RNA (5'-R(P*CP*AP*AP*GP*CP*CP*GP*AP*GP*GP*AP*GP*AP*G)-3')
  • Ligand: ZINC ION
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
Function / homology
Function and homology information


MDA-5 signaling pathway / Ub-specific processing proteases / positive regulation of response to cytokine stimulus / pattern recognition receptor activity / negative regulation of viral genome replication / type I interferon-mediated signaling pathway / cellular response to exogenous dsRNA / antiviral innate immune response / protein sumoylation / positive regulation of interferon-alpha production ...MDA-5 signaling pathway / Ub-specific processing proteases / positive regulation of response to cytokine stimulus / pattern recognition receptor activity / negative regulation of viral genome replication / type I interferon-mediated signaling pathway / cellular response to exogenous dsRNA / antiviral innate immune response / protein sumoylation / positive regulation of interferon-alpha production / ribonucleoprotein complex binding / positive regulation of interferon-beta production / response to virus / cellular response to virus / positive regulation of interleukin-6 production / positive regulation of tumor necrosis factor production / double-stranded RNA binding / protein complex oligomerization / defense response to virus / RNA helicase activity / single-stranded RNA binding / RNA helicase / protein domain specific binding / innate immune response / ATP hydrolysis activity / mitochondrion / DNA binding / zinc ion binding / ATP binding / identical protein binding / nucleus / cytoplasm
Similarity search - Function
RIG-I-like receptor, C-terminal / RIG-I receptor C-terminal domain / RIG-I-like receptor, C-terminal regulatory domain / RIG-I-like receptor, C-terminal domain superfamily / C-terminal domain of RIG-I / RIG-I-like receptor (RLR) C-terminal regulatory (CTR) domain profile. / Caspase recruitment domain / Caspase recruitment domain / Helicase/UvrB, N-terminal / Type III restriction enzyme, res subunit ...RIG-I-like receptor, C-terminal / RIG-I receptor C-terminal domain / RIG-I-like receptor, C-terminal regulatory domain / RIG-I-like receptor, C-terminal domain superfamily / C-terminal domain of RIG-I / RIG-I-like receptor (RLR) C-terminal regulatory (CTR) domain profile. / Caspase recruitment domain / Caspase recruitment domain / Helicase/UvrB, N-terminal / Type III restriction enzyme, res subunit / Death-like domain superfamily / Helicase conserved C-terminal domain / helicase superfamily c-terminal domain / Superfamilies 1 and 2 helicase C-terminal domain profile. / Superfamilies 1 and 2 helicase ATP-binding type-1 domain profile. / DEAD-like helicases superfamily / Helicase, C-terminal / Helicase superfamily 1/2, ATP-binding domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Interferon-induced helicase C domain-containing protein 1
Similarity search - Component
Biological speciesMus musculus (house mouse) / Pseudomonas virus phi6 (bacteriophage)
Methodhelical reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsSingh R / Herrero del Valle A / Yu Q / Modis Y
Funding support United Kingdom, 2 items
OrganizationGrant numberCountry
Wellcome Trust217191/Z/19/Z United Kingdom
Wellcome Trust101908/Z/13/Z United Kingdom
CitationJournal: Nat Commun / Year: 2021
Title: MDA5 disease variant M854K prevents ATP-dependent structural discrimination of viral and cellular RNA.
Authors: Qin Yu / Alba Herrero Del Valle / Rahul Singh / Yorgo Modis /
Abstract: Our innate immune responses to viral RNA are vital defenses. Long cytosolic double-stranded RNA (dsRNA) is recognized by MDA5. The ATPase activity of MDA5 contributes to its dsRNA binding selectivity. ...Our innate immune responses to viral RNA are vital defenses. Long cytosolic double-stranded RNA (dsRNA) is recognized by MDA5. The ATPase activity of MDA5 contributes to its dsRNA binding selectivity. Mutations that reduce RNA selectivity can cause autoinflammatory disease. Here, we show how the disease-associated MDA5 variant M854K perturbs MDA5-dsRNA recognition. M854K MDA5 constitutively activates interferon signaling in the absence of exogenous RNA. M854K MDA5 lacks ATPase activity and binds more stably to synthetic Alu:Alu dsRNA. CryoEM structures of MDA5-dsRNA filaments at different stages of ATP hydrolysis show that the K854 sidechain forms polar bonds that constrain the conformation of MDA5 subdomains, disrupting key steps in the ATPase cycle- RNA footprint expansion and helical twist modulation. The M854K mutation inhibits ATP-dependent RNA proofreading via an allosteric mechanism, allowing MDA5 to form signaling complexes on endogenous RNAs. This work provides insights on how MDA5 recognizes dsRNA in health and disease.
History
DepositionJan 16, 2021-
Header (metadata) releaseNov 17, 2021-
Map releaseNov 17, 2021-
UpdateDec 1, 2021-
Current statusDec 1, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.015
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.015
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  • Surface view with fitted model
  • Atomic models: PDB-7bkp
  • Surface level: 0.015
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7bkp
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_12213.png

Downloads & links

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Map

FileDownload / File: emd_12213.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.86 Å
Density
Contour LevelBy AUTHOR: 0.015 / Movie #1: 0.015
Minimum - Maximum-0.044178158 - 0.116851114
Average (Standard dev.)0.00025842924 (±0.002447467)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 240.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.860.860.86
M x/y/z280280280
origin x/y/z0.0000.0000.000
length x/y/z240.800240.800240.800
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS280280280
D min/max/mean-0.0440.1170.000

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Supplemental data

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Mask #1

Fileemd_12213_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_12213_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_12213_half_map_2.map
Projections & Slices
AxesZYX

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Histogram (log scale)

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Sample components

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Entire : M854K MDA5-dsRNA filament in complex with ATP

EntireName: M854K MDA5-dsRNA filament in complex with ATP
Components
  • Complex: M854K MDA5-dsRNA filament in complex with ATP
    • Complex: INTERFERON-INDUCED HELICASE C DOMAIN-CONTAINING PROTEIN
      • Protein or peptide: Interferon-induced helicase C domain-containing protein 1
    • Complex: RNA
      • RNA: RNA (5'-R(P*CP*UP*CP*UP*CP*CP*UP*CP*GP*GP*CP*UP*UP*G)-3')
      • RNA: RNA (5'-R(P*CP*AP*AP*GP*CP*CP*GP*AP*GP*GP*AP*GP*AP*G)-3')
  • Ligand: ZINC ION
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION

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Supramolecule #1: M854K MDA5-dsRNA filament in complex with ATP

SupramoleculeName: M854K MDA5-dsRNA filament in complex with ATP / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Molecular weightExperimental: 1.91 kDa/nm

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Supramolecule #2: INTERFERON-INDUCED HELICASE C DOMAIN-CONTAINING PROTEIN

SupramoleculeName: INTERFERON-INDUCED HELICASE C DOMAIN-CONTAINING PROTEIN
type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Mus musculus (house mouse)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)

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Supramolecule #3: RNA

SupramoleculeName: RNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#3
Source (natural)Organism: Pseudomonas virus phi6 (bacteriophage)
Recombinant expressionOrganism: synthetic construct (others)

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Macromolecule #1: Interferon-induced helicase C domain-containing protein 1

MacromoleculeName: Interferon-induced helicase C domain-containing protein 1
type: protein_or_peptide / ID: 1 / Details: ATP / Number of copies: 1 / Enantiomer: LEVO / EC number: RNA helicase
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 116.122359 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MSIVCSAEDS FRNLILFFRP RLKMYIQVEP VLDHLIFLSA ETKEQILKKI NTCGNTSAAE LLLSTLEQGQ WPLGWTQMFV EALEHSGNP LAARYVKPTL TDLPSPSSET AHDECLHLLT LLQPTLVDKL LINDVLDTCF EKGLLTVEDR NRISAAGNSG N ESGVRELL ...String:
MSIVCSAEDS FRNLILFFRP RLKMYIQVEP VLDHLIFLSA ETKEQILKKI NTCGNTSAAE LLLSTLEQGQ WPLGWTQMFV EALEHSGNP LAARYVKPTL TDLPSPSSET AHDECLHLLT LLQPTLVDKL LINDVLDTCF EKGLLTVEDR NRISAAGNSG N ESGVRELL RRIVQKENWF STFLDVLRQT GNDALFQELT GGGCPEDNTD LANSSHRDGP AANECLLPAV DESSLETEAW NV DDILPEA SCTDSSVTTE SDTSLAEGSV SCFDESLGHN SNMGRDSGTM GSDSDESVIQ TKRVSPEPEL QLRPYQMEVA QPA LDGKNI IICLPTGSGK TRVAVYITKD HLDKKKQASE SGKVIVLVNK VMLAEQLFRK EFNPYLKKWY RIIGLSGDTQ LKIS FPEVV KSYDVIISTA QILENSLLNL ESGDDDGVQL SDFSLIIIDE CHHTNKEAVY NNIMRRYLKQ KLRNNDLKKQ NKPAI PLPQ ILGLTASPGV GAAKKQSEAE KHILNICANL DAFTIKTVKE NLGQLKHQIK EPCKKFVIAD DTRENPFKEK LLEIMA SIQ TYCQKSPMSD FGTQHYEQWA IQMEKKAAKD GNRKDRVCAE HLRKYNEALQ INDTIRMIDA YSHLETFYTD EKEKKFA VL NDSDKSDDEA SSCNDQLKGD VKKSLKLDET DEFLMNLFFD NKKMLKKLAE NPKYENEKLI KLRNTILEQF TRSEESSR G IIFTKTRQST YALSQWIMEN AKFAEVGVKA HHLIGAGHSS EVKPMTQTEQ KEVISKFRTG EINLLIATTV AEEGLDIKE CNIVIRYGLV TNEIAMVQAR GRARADESTY VLVTSSGSGV TEREIVNDFR EKMKYKAINR VQNMKPEEYA HKILELQVQS ILEKKMKVK RSIAKQYNDN PSLITLLCKN CSMLVCSGEN IHVIEKMHHV NMTPEFKGLY IVRENKALQK KFADYQTNGE I ICKCGQAW GTMMVHKGLD LPCLKIRNFV VNFKNNSPKK QYKKWVELPI RFPDLDYSEY CLYSDED

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Macromolecule #2: RNA (5'-R(P*CP*UP*CP*UP*CP*CP*UP*CP*GP*GP*CP*UP*UP*G)-3')

MacromoleculeName: RNA (5'-R(P*CP*UP*CP*UP*CP*CP*UP*CP*GP*GP*CP*UP*UP*G)-3')
type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Pseudomonas virus phi6 (bacteriophage)
Molecular weightTheoretical: 4.35258 KDa
SequenceString:
CUCUCCUCGG CUUG

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Macromolecule #3: RNA (5'-R(P*CP*AP*AP*GP*CP*CP*GP*AP*GP*GP*AP*GP*AP*G)-3')

MacromoleculeName: RNA (5'-R(P*CP*AP*AP*GP*CP*CP*GP*AP*GP*GP*AP*GP*AP*G)-3')
type: rna / ID: 3 / Number of copies: 1
Source (natural)Organism: Pseudomonas virus phi6 (bacteriophage)
Molecular weightTheoretical: 4.587852 KDa
SequenceString:
CAAGCCGAGG AGAG

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Macromolecule #4: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 4 / Number of copies: 1 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #5: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 5 / Number of copies: 1 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM

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Macromolecule #6: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 6 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

Concentration1 mg/mL
BufferpH: 7.8
Component:
ConcentrationNameFormula
30.0 mMHEPES
0.15 MKCl
1.0 mMDithiothreitol
10.0 mMATP
GridModel: UltrAuFoil / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Details: machine step 6
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 277.2 K / Instrument: FEI VITROBOT MARK IV
Details: Grids were blotted for 3 s; blot force 10, 5, 0, -5, -10.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsPhase plate: OTHER / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number real images: 7147 / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated defocus max: -4.0 µm / Calibrated defocus min: -0.25 µm / Illumination mode: FLOOD BEAM / Imaging mode: DARK FIELD / Cs: 2.7 mm / Nominal defocus max: -2.5 µm / Nominal defocus min: -1.2 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsThe images were motion-corrected
Final reconstructionNumber classes used: 1
Applied symmetry - Helical parameters - Δz: 43.92 Å
Applied symmetry - Helical parameters - Δ&Phi: 74.63 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Resolution.type: BY AUTHOR / Resolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 383128
CTF correctionSoftware - Name: CTFFIND
Segment selectionNumber selected: 1068108 / Software - Name: RELION (ver. 3.1)
Startup modelType of model: EMDB MAP
EMDB ID:

4338

Final angle assignmentType: NOT APPLICABLE / Software - Name: RELION (ver. 3.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6g19

RefinementSpace: RECIPROCAL / Protocol: OTHER / Overall B value: 50 / Target criteria: Correlation coefficient
Output model

PDB-7bkp:
CryoEM structure of disease related M854K MDA5-dsRNA filament in complex with ATP

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